Clinical and research investigations in the various fields of biotechnology frequently involve the identification of the components of samples of biological mixtures, the components ranging from whole organisms to fragments of nucleic acids and proteins. The component species of a mixture are identified by any of a variety of separation procedures, prominent among which are electrophoresis, size exclusion chromatography, and isoelectric focusing. The sample is typically dissolved or suspended in an aqueous buffer solution, and the medium on which the separation is performed is typically a porous substrate such as a polyacrylamide or agarose gel. Depending on the experiment being performed, the sample either remains in the gel or is transferred to the surface of a membranous support, commonly known as a “blot,” to allow greater accessibility. Since the separated species are often undetectable by themselves, they are associated for detection purposes with detectable moieties such as light-absorptive, radioactive, luminescent, or fluorescent reporter moieties. These reporter moieties are either covalently bound to the species prior to the separation or applied after the separation as general affinity stains or as biologically based molecule-specific probes. Procedures that include the attachment of reporter moieties to separated species are variously termed “Southern,” “Northern,” and “Western” blotting.
For chromatographically separated species, whether the separation has been performed in either one dimension or two dimensions, detection methods of choice have historically been those that generate an image of the array of separated species. Such images have traditionally been recorded on x-ray film or on photographic negatives and prints. Digital imaging has since been developed and has grown in popularity in recent years as digital technology has improved and become more accessible to the scientific community. At present, the most common imaging techniques are those involving the use of charge coupled devices (CCDs). CCDs are particularly well suited to the imaging of chromatographic arrays because of the sensitivity of CCDs in both the visible and near-infrared spectra, where the most biological sample detections occur.
A digital imaging instrument generally combines a CCD camera with a light source(s) to illuminate the biological sample, with both the CCD camera and the light source(s) retained in an enclosure that is sealed against ambient light. Depending on its design, the instrument may contain or implement light sources that emit excitation light at specific wavelength bands and yet include a range of emission filters that allow the detection of specific reporter moieties to be optimized by selecting the most appropriate filter.